Acute myocardial infarction detection kit taking S100A12 as marker and detection method

Abstract

The invention provides an acute myocardial infarction detection kit taking S100A12 as a marker and a detection method, and belongs to the technical field of biomedicine. The invention provides an immunochromatography detection kit for detecting an S100A12 antigen. The immunochromatography detection kit comprises a reconstitution fluid, a quantum dot labeled antibody conjugate and an immunochromatography detection test strip which are independently packaged, wherein an antibody in the quantum dot labeled antibody conjugate is an S100A12 specific monoclonal antibody, and a detection line of the immunochromatography detection test strip is coated with an S100A12 capture antibody. The S100A12 protein is used as an acute myocardial infarction attack extremely-early diagnosis marker, a result can be displayed at high sensitivity within a short time after a sample is added, the detection limit can reach 2.5 pg / mL, and quantitative detection of an S100A12 low-concentration sample in blood of an acute myocardial infarction patient at the early attack stage is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to an acute myocardial infarction detection kit and detection method taking S100A12 as a marker. BACKGROUND

[0002] Acute myocardial infarction is one of common diseases that seriously threaten human health. The pathogenesis of acute myocardial infarction is that the rupture of coronary atherosclerotic plaque and thrombosis lead to acute injury and necrosis of myocardial cells, and about 90% to 95% of transmural myocardial infarction is caused by local thrombosis due to the rupture of coronary atherosclerotic plaque. The mechanism is that various cardiovascular risk factors cause endothelial injury and dysfunction, leading to chronic inflammation of the vascular wall, and macrophages, T lymphocytes, B lymphocytes, smooth muscle cells and various inflammatory factors can further promote and amplify the chronic inflammation of the vascular wall, causing the progression of atherosclerosis, the formation and rupture of unstable plaques, the thrombosis in the coronary artery and the transmural necrosis of myocardial tissue, and finally the occurrence of acute myocardial infarction.

[0003] The existing rapid detection of acute myocardial infarction mainly relies on traditional markers such as troponin (cTnI / T) and myoglobin (MYO), which generally requires long-term laboratory detection, or uses the traditional colloidal gold immunochromatography method for detection, although the time is shortened, but the sensitivity is low and cannot be quantified, which cannot meet the bedside detection. SUMMARY

[0004] The application provides an acute myocardial infarction detection kit and detection method taking S100A12 as a marker, which can detect the content of S100A12 in the very early stage of acute myocardial infarction with high sensitivity, and can meet the bedside detection.

[0005] The application provides an antigen immunochromatographic detection kit for detecting S100A12, which comprises a reconstitution solution, a quantum dot-labeled antibody conjugate and an immunochromatographic test strip packaged independently.

[0006] The antibody in the quantum dot-labeled antibody conjugate is a specific monoclonal antibody of S100A12, and a S100A12 capture antibody is coated on the detection line of the immunochromatographic test strip.

[0007] In a preferred mode of the application, the reconstitution solution contains Tris-HCl, PEG-2000, Tween-20 and sucrose.

[0008] In a preferred mode of the application, the reconstitution solution takes Tris-HCl as a solvent and contains the following ingredients with mass percentage: 0.5% to 2% PEG-20000, 0.25% to 1.5% Tween-20 and 1% to 6% sucrose.

[0009] In a preferred embodiment of the present invention, the quantum dot-labeled antibody conjugate comprises a quantum dot-labeled antibody conjugate QBs-Ab1 prepared by coupling carboxylated quantum dot microspheres with the S100A12-specific monoclonal antibody Ab1.

[0010] In a preferred embodiment of the present invention, the structure of the immunochromatographic test strip comprises a sample pad, an NC membrane and absorbent paper assembled together on a bottom plate.

[0011] In a preferred embodiment of the present invention, a detection line and a quality control line are set on the NC membrane, and the concentration of the S100A12 capture antibody coated on the detection line is 0.5-1.5 mg / mL;

[0012] The quality control line was coated with mouse anti-rabbit IgG at a concentration of 0.5-1.5 mg / mL.

[0013] The present invention also provides a method for preparing the antigen immunochromatographic detection kit, which comprises combining the complex solution, the quantum dot-labeled antibody conjugate and the immunochromatographic detection test strip.

[0014] The present invention also provides the use of the antigen immunochromatographic detection kit in preparing an early diagnosis reagent for acute myocardial infarction.

[0015] The present invention also provides a method for detecting S100A12 for purposes other than disease diagnosis and / or treatment. When the above antigen immunochromatographic detection kit is used for detection, the method comprises the following steps: diluting the quantum dot-labeled antibody conjugate with a reconstitution solution, mixing and incubating the mixture with a sample to be tested to obtain an incubation solution;

[0016] The incubation solution is spotted onto an immunochromatographic test strip, and the content of S100A12 in the sample to be tested is calculated based on the signal intensity.

[0017] In a preferred embodiment of the present invention, the incubation solution is diluted 1000 to 2500 times before the spotting.

[0018] In a preferred embodiment of the present invention, the relationship between the signal intensity and the concentration of S100A12 in the sample includes: y = 183.3 + (11389 - 183.3) / (1 + 10 (2.847-x)×1.167 ), R 2 =0.9942, x is the Log value of the S100A12 concentration in the sample, the unit of the S100A12 concentration in the sample is pg / mL, and y is the fluorescence signal value.

[0019] Beneficial effects: the S100A12 protein is used as an acute myocardial infarction very early diagnosis marker, which can be detected within 30 minutes of onset. The antigen immunochromatographic detection kit for detecting S100A12 protein with high sensitivity is developed, which includes independently packaged reconstitution solution, quantum dot labeled antibody conjugate and immunochromatographic detection test strip; wherein the antibody in the quantum dot labeled antibody conjugate is a specific monoclonal antibody of S100A12, and the S100A12 capture antibody is coated on the detection line of the immunochromatographic detection test strip.

[0020] When the method is used for detecting S100A12 protein or evaluating acute myocardial infarction very early, first, the quantum dots are coupled with the S100A12 specific monoclonal antibody Ab1 to prepare the quantum dot labeled specific antibody conjugate QBs-Ab1; the S100A12 capture antibody Ab2 (T line) and the mouse anti-rabbit IgG (C line) are coated on the NC membrane. When the detection is performed, after the sample containing S100A12 is mixed with the diluted conjugate, Ab1 is combined with S100A12 in the sample to form a QBs-Ab1-S100A12 complex; the complex is dropped on the sample pad and chromatographed upwards to the T line, the capture antibody is combined with S100A12 in the complex to form a QBs-Ab1-S100A12-Ab2 complex deposited on the T line; the remaining QBs-Ab1-S100A12 complex continues to chromatograph upwards, and Ab1 in the complex reacts with the mouse anti-rabbit IgG to be deposited on the C line. After 15 minutes of sample addition, the signal value is detected by using a fluorescence immunoassay instrument, and S100A12 can be quantified according to the standard curve. If the T line signal value of the sample detection is greater than 11389, it indicates that the sample concentration is too high, and the sample needs to be diluted and detected again, otherwise the accuracy of the quantitative result will be affected; if the T line signal value is less than 183.3, it indicates that the sample concentration is too low, and the original sample needs to be diluted and detected again. The kit and the detection method can improve the signal strength by using the fluorescent quantum dot microspheres as the immunolabeling agent, the detection limit can reach 2.5 pg / mL, and the quantitative detection of the low concentration sample of S100A12 in the blood of the patient with acute myocardial infarction in the early stage of onset is realized. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The structure of the immunochromatographic detection test strip is shown in the figure, wherein 1-4 represent the sample pad, the water absorption paper, the nitrocellulose (NC) membrane and the bottom plate respectively;

[0022] Figure 2 It is a T line concentration and conjugate dilution multiple screening result graph;

[0023] Figure 3 It is a standard curve;

[0024] Figure 4For specificity detection result figure;

[0025] Figure 5 For precision detection result figure. DETAILED DESCRIPTION

[0026] The application provides an antigen immunochromatographic detection kit for detecting S100A12, which comprises a reconstitution solution, a quantum dot-labeled antibody conjugate and an immunochromatographic detection test strip packaged independently.

[0027] The antibody in the quantum dot-labeled antibody conjugate is a specific single antibody of S100A12, and a S100A12 capture antibody is coated on a detection line of the immunochromatographic detection test strip.

[0028] The content of taking S100A12 as an early diagnosis marker for acute myocardial infarction in the application has been disclosed in Chinese Patent CN107271681A and is fully introduced into the application by reference.

[0029] The reconstitution solution in the kit contains Tris-HCl as a solvent and contains Tris-HCl, PEG-20000, Tween-20 and sucrose, and the mass percentage of PEG-20000 can be 0.5% to 2%, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%, and in one embodiment, 1% is used for experiment.

[0030] The PEG-20000 can increase the viscosity of the sample liquid to prevent protein precipitation, and its macromolecular structure can occupy the non-specific binding sites on the membrane surface to reduce the non-specific adsorption of antibodies or antigens. At the same time, it can adjust the physical properties of the sample liquid, such as viscosity and osmotic pressure, so as to provide a more stable environment for the immune reaction.

[0031] The reconstitution solution further contains Tween-20, and the mass percentage of Tween-20 can be 0.25% to 1.5%, such as 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%, and in one embodiment, 0.5% is used for experiment. The Tween-20 in the application can block the unbound sites on the membrane surface to reduce the non-specific adsorption of antibodies or antigens, and can also prevent protein denaturation or aggregation during the detection process, thereby improving the sensitivity of the detection.

[0032] The complex solution also contains sucrose, which can account for 1% to 6% by mass percentage, such as 1%, 2%, 3%, 4%, 5% or 6%, and in one embodiment, 3% is used for experiments. The sucrose in the application is mainly used to stabilize the protein structure, prevent protein denaturation or aggregation during detection by maintaining high osmotic pressure of the solution. At the same time, it can reduce the non-specific binding of proteins in the sample to the membrane surface, thereby improving the specificity and stability of the detection.

[0033] In the embodiments of the application, it is confirmed that the order of the influence of each component in the complex solution on the signal value is: sucrose > pH > Tween-20 > PEG-20000, and the best complex solution ratio screened is: pH 7.5 Tris-HCl, containing 1wt% PEG-2000, 0.5wt% Tween-20, 3wt% sucrose.

[0034] The kit comprises a quantum dot-labeled antibody conjugate, which in specific embodiments can be a quantum dot-labeled antibody conjugate QBs-Ab1 prepared by coupling carboxylated quantum dot microspheres with S100A12-specific monoclonal antibody Ab1. In one embodiment of the present application, the coupling is completed by using an activated ester method, and carboxylated quantum dot microspheres (QBs) are coupled with S100A12-specific monoclonal antibody Ab1 to prepare quantum dot-labeled specific antibody conjugate QBs-Ab1. The S100A12-specific monoclonal antibody Ab1 used in one embodiment of the present application is purchased from BioSun, and its product number is BSP0123C. The QBs used in one embodiment of the present application are water-soluble carboxylated CdSe / ZnS quantum dot microspheres (model number: FM610C; particle size: 120 nm; batch number: QBB12120D17586M) purchased from Beijing Nanjing Biotechnology Co., Ltd. The preparation of the quantum dot-labeled specific antibody conjugate QBs-Ab1 comprises the following steps: 25 μL of quantum dot microspheres (QBs, 1 μmol / L) is mixed with 25 μL of MBS (20 mmol / L, pH 6.0), 1 μL of 20 mg / mL EDC and NHS solution prepared with DMSO is added, and vortexed, 37°C, light-avoiding activation for 15 min; centrifuged at 10000 rcf for 20 min, the supernatant is discarded, 25 μL of MBS (10 mmol / L, pH 6.0) is added to resuspend the precipitate, and vortexed; 10 μg of antibody Ab1 is added and vortexed, 37°C, 800 rpm, light-avoiding shaking incubation for 1 h; 25 μL of 10% BSA solution is added and vortexed, 37°C, light-avoiding blocking for 30 min; centrifuged at 10000 rcf for 15 min, the supernatant is discarded, 50 μL of boric acid buffer (5 mmol / L, pH 8.0) + 1% BSA solution is added to resuspend and wash once, centrifuged, and the supernatant is discarded; 25 μL of boric acid buffer (5 mmol / L, pH 8.0) + 1% BSA solution is added to resuspend, and stored at 4°C for standby.

[0035] The kit comprises an immunochromatographic test strip, which has a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a test line and a control line. Figure 1The structure shown includes a sample pad, a conjugate pad, a NC membrane, and absorbent paper assembled together on a base plate. A detection line (T line) and a quality control line (C line) are provided on the NC membrane. The T line is coated with the S100A12 capture antibody Ab2. The concentration of the S100A12 capture antibody Ab2 can be 0.5 to 1.5 mg / mL, such as 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, or 1.5 mg / mL. The S100A12 capture antibody Ab2 used in one embodiment of the present invention was purchased from Biosun, with the product number BSP0123D. The present invention is coated with mouse anti-rabbit IgG on the C line at a concentration of 0.5 to 1.5 mg / mL, such as 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL or 1.5 mg / mL.

[0036] The present invention also provides a method for preparing the antigen immunochromatographic detection kit, which comprises combining the complex solution, the quantum dot-labeled antibody conjugate and the immunochromatographic detection test strip.

[0037] The preparation method of the kit of the present invention is simple, and only requires assembling the solutions, test materials, etc.

[0038] The present invention also provides the use of the antigen immunochromatographic detection kit in preparing an early diagnosis reagent for acute myocardial infarction.

[0039] The present invention uses S100A12 protein in the blood as a predictive marker for acute myocardial infarction, which can be detected within 30 minutes of onset and can be applied to the early detection of acute myocardial infarction.

[0040] The present invention also provides a method for detecting S100A12 for purposes other than disease diagnosis and / or treatment. When the above antigen immunochromatographic detection kit is used for detection, the method comprises the following steps: dissolving the quantum dot-labeled antibody conjugate in a reconstitution solution, mixing and incubating the solution with a sample to be tested to obtain an incubation solution;

[0041] The incubation solution is spotted onto an immunochromatographic test strip, and the content of S100A12 in the sample to be tested is calculated based on the signal intensity.

[0042] The QBs-Ab1 diluted by the reconstitution solution is first mixed with the sample to be tested and incubated. Since the concentration of QBs-Ab1 is high, it is usually diluted by about 2000 times before use. During the test, the QBs is first diluted by 150 times by using the reconstitution solution, then mixed with the plasma sample (previously diluted by 100-500 times by using a sample diluent such as PB solution) and the reconstitution solution in a volume ratio of 1:1:8, and then incubated and loaded. The final dilution ratio of the conjugate is 1000-2500 times.

[0043] The sample to be tested in the present application can be plasma. The sample to be tested is mixed with the diluted conjugate and the reconstitution solution for incubation. In one embodiment, the incubation is carried out at 37°C and 800 rpm for 5 minutes.

[0044] In the present application, when the sample containing S100A12 is mixed with the conjugate diluted by the loading solution, Ab1 binds to S100A12 in the sample to form a QBs-Ab1-S100A12 complex. When the complex is dropped on the sample pad and chromatographed upwards to the T line, the capture antibody binds to S100A12 in the complex to form a QBs-Ab1-S100A12-Ab2 complex deposited on the T line. The remaining QBs-Ab1-S100A12 complex continues to chromatograph upwards, and Ab1 in the complex reacts with the mouse anti-rabbit IgG to be deposited on the C line. After 15 minutes of loading, the S100A12 can be quantified by binding to a fluorescence immunoassay instrument. In one embodiment of the present application, the relationship between the signal intensity and the concentration of S100A12 in the sample includes: y = 183.3 + (11389-183.3) / (1+10 (2.847 -x)×1.167 ), R 2 = 0.9942, x is the Log value of the concentration of S100A12 in the sample, and the unit of the concentration of S100A12 in the sample is pg / mL, and y is the fluorescence signal value. The signal value is detected by using a fluorescence immunoassay instrument, and the S100A12 can be quantified according to the standard curve. If the T line signal value of the sample detection is greater than 11389, it means that the sample concentration is too high, and it needs to be diluted further for re-detection, otherwise it will affect the accuracy of the quantitative result. If the T line signal value is less than 183.3, it means that the sample concentration is too low, and the original sample needs to be diluted at a low ratio for re-detection.

[0045] In order to further illustrate the present application, a kind of acute myocardial infarction detection kit and detection method provided by the present application with S100A12 as a marker are described in detail in conjunction with the embodiments below, but they cannot be understood as limiting the scope of protection of the present application.

[0046] Example 1

[0047] 1.1 Preparation of antibody conjugate

[0048] Take 25 μL of quantum dot microspheres (QBs, 1 μmol / L) and mix with 25 μL of M ES (20 mmol / L, pH 6.0), add 1 μL of 20 mg / mL EDC and NHS solution prepared with DMSO, vortex mix, activate at 37°C in the dark for 15 min; centrifuge at 10000 rcf for 20 min, discard the supernatant, resuspend the precipitate with 25 μL of M ES (10 mmol / L, pH 6.0), vortex mix; add 10 μg of antibody Ab1, vortex mix, incubate at 37°C, 800 rpm, in the dark for 1 h; add 25 μL of 10% BSA solution, vortex mix, block at 37°C in the dark for 30 min; centrifuge at 10000 rcf for 15 min, discard the supernatant, resuspend in 50 μL of boric acid buffer (5 mmol / L, pH 8.0) + 1% BSA solution, centrifuge, discard the supernatant; resuspend in 25 μL of boric acid buffer (5 mmol / L, pH 8.0) + 1% BSA solution to obtain the antibody conjugate, store at 4°C for later use.

[0049] 1.2 Assembly of test paper

[0050] Paste the nitrocellulose membrane (NC membrane) on the bottom plate, use the automatic gold spraying membrane drawing instrument to coat the S100A12 capture antibody Ab2 (diluted to 0.8 mg / mL) on the NC membrane as the test line (T line), and coat the mouse anti-rabbit IgG polyclonal antibody (diluted to 0.5 mg / mL) on the NC membrane as the control line (C line). After drawing the membrane, place the plate in an oven at 37°C to dry for 1 h; assemble the sample pad and absorbent paper on the bottom plate, cut into 3 mm wide strips using the numerical control strip cutting machine, dry, and store in the dark for later use.

[0051] Example 2

[0052] 2.1 Optimization of sample loading solution components

[0053] The size and content of each component of the sample loading solution have a great influence on the detection signal intensity and background value. The optimal reconstitution solution ratio was obtained by optimizing the main influencing factors of the reconstitution solution, i.e. pH value, content of sucrose, PEG-20000, and Tween-20. The specific orthogonal test scheme is shown in Table 1. According to the detection results, the signal value influence ranking is sucrose > pH > Tween-20 > PEG-20000, and the optimal reconstitution solution ratio is pH 7.5 Tris-HCl, containing 1% PEG-2000, 0.5% Tween-20, and 3% sucrose.

[0054] Table 1. Orthogonal experiment of reconstitution solution and results

[0055]

[0056] 2.2T line concentration and conjugate dilution multiple screening

[0057] T-line antibody concentrations were set at 0.5, 0.8, and 1.0 mg / mL, and conjugate dilutions were set at 1000, 1500, 2000, and 2500. S100A12 antigen was detected using test strips. Twelve experiments were set up, as shown below:

[0058] Experiments 1-3: The conjugate was diluted 1000-fold, and the T-line concentrations were 0.5, 0.8, and 1.0 mg / mL, respectively;

[0059] Experiments 4–6: The conjugate was diluted 1500-fold, and the T-line concentrations were 0.5, 0.8, and 1.0 mg / mL, respectively;

[0060] Experiments 7–9: The conjugate was diluted 2000-fold, and the T-line concentrations were 0.5, 0.8, and 1.0 mg / mL, respectively;

[0061] Experiments 10-12: The conjugate was diluted 2500-fold, and the T-line concentrations were 0.5, 0.8, and 1.0 mg / mL, respectively.

[0062] Test results such as Figure 2 As shown in the figure, at each dilution factor, the signal value increases with increasing T-line concentration, and the increase at 0.8 mg / mL is greater than that at 1.0 mg / mL. Therefore, a T-line concentration of 0.8 mg / mL can be selected for subsequent dilutions. At the same T-line concentration, the signal value increases with decreasing conjugate dilution factor, and the increase is greatest at a dilution factor of 1500. Therefore, a 1500-fold dilution factor is selected for subsequent conjugate dilutions.

[0063] Example 3

[0064] 3.1 Construction of the standard track

[0065] Plasma samples from patients with acute myocardial infarction (according to the ELISA kit quantitative results, the sample concentration was 507.522 ng / mL) were tested using the test strips prepared in Examples 1 and 2. The samples were diluted to 5000, 2500, 1000, 500, 250, 100, 25, 10, and 2.5 pg / mL. A standard curve was plotted based on the detected signal values.

[0066] The results are as follows Figure 3 As shown, the curve equation is: y = 183.3 + (11389-183.3) / (1 + 10 ((2.847-x)×1.167) ), R 2 =0.9942. The calculated LOD was 2.5 pg / mL, of which the visualized LOD was 10 pg / mL.

[0067] 3.2 Positive reference sample compliance rate

[0068] The positive reference samples (P1-P6) were detected, and each sample was detected three times. 18 μL of each positive reference sample P1-P6 was mixed with 162 μL of sample diluent (containing 150-fold diluted antibody conjugate), incubated at 37°C for 5 min, and then detected, signal collected, and analyzed. The results are shown in Table 2, and the recovery rate of the quantitative results was between 92.537% and 108.793%.

[0069] Table 2 Detection results of positive reference samples

[0070]

[0071] 3.3 Sensitivity

[0072] The detection limit reference samples L1 and L2 were detected, and each sample was detected three times. 18 μL of each detection limit reference sample L1 and L2 was mixed with 162 μL of sample diluent (containing 150-fold diluted antibody conjugate), incubated at 37°C for 5 min, and then detected, signal collected, and analyzed. The results are shown in Table 3, and the minimum detection limit of the reagent was 2.5 pg / mL, and the recovery rate of the quantitative results of the detection limit reference samples was between 90% and 110%.

[0073] Table 3 Detection results of detection limit reference samples

[0074]

[0075] 3.4 Specificity

[0076] The negative reference samples N1-N3 were detected, and each sample was detected three times. 18 μL of each negative reference sample (N1-N3) was mixed with 162 μL of sample diluent (containing 150-fold diluted antibody conjugate), incubated at 37°C for 5 min, and then detected, signal collected, and analyzed. The results are shown in Table 5, and all were negative, with a fluorescence signal value of less than 85 a.u. Figure 4

[0077] 3.5 Precision

[0078] The precision reference sample R was detected 10 times (Table 4). 60 μL of the precision reference sample R was mixed with 540 μL of sample diluent (containing 150-fold diluted antibody conjugate), incubated for 5 min, and then detected, signal collected, and analyzed. The results are shown in Table 4, and the CV was ≤5.7%. Figure 5

[0079] Table 4 Reference sample information

[0080] ​​

[0081] Example 4

[0082] 4.1 Quantitative detection of plasma samples of acute myocardial infarction patients

[0083] Quantitative detection was performed on 25 plasma samples of acute myocardial infarction patients using ELISA kit (S100A12 / EN-RAGE ELISA Kit Ver.2, MBL). Meanwhile, the 25 plasma samples were detected using the test paper prepared according to the optimal parameters of the present application, and the sample concentrations were calculated according to the standard curve in Example 3. The results are shown in Table 5. It can be seen that the quantitative concentration of the prepared test paper for the plasma samples of acute myocardial infarction patients is basically consistent with the quantitative results of the ELISA kit, and the recovery rate is 90.05% to 108.66%.

[0084] Table 5 Quantitative results of test paper

[0085]

[0086] 4.2 Quantitative detection of control samples

[0087] The concentration of the control samples detected by S100A12 Elisa kit was lower than 100 ng / mL, and the quantitative detection was performed on three of the samples using the test paper strip according to the present application, and the concentrations were 36.820, 95.039 and 69.414 ng / mL, respectively.

[0088] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. An immunochromatographic detection kit for detecting S100A12 antigen, characterized in that: Includes independently packaged reconstitution solution, quantum dot-labeled antibody conjugate and immunochromatographic test strips; The antibody in the quantum dot-labeled antibody conjugate is a specific monoclonal antibody for S100A12, and the detection line of the immunochromatographic test strip is coated with a S100A12 capture antibody.

2. The antigen immunochromatographic detection kit according to claim 1, characterized in that: The reconstitution solution contains Tris-HCl, PEG-20000, Tween-20 and sucrose.

3. The antigen immunochromatographic detection kit according to claim 2, characterized in that: The complex solution uses Tris-HCl as a solvent and contains the following components in percentage by weight: 0.5% to 2% PEG-20000, 0.25% to 1.5% Tween-20 and 1% to 6% sucrose.

4. The antigen immunochromatographic detection kit according to claim 1, characterized in that: The quantum dot-labeled antibody conjugate includes the quantum dot-labeled antibody conjugate QBs-Ab1 prepared by coupling carboxylated quantum dot microspheres with the S100A12-specific monoclonal antibody Ab1.

5. The antigen immunochromatographic detection kit according to claim 1, characterized in that: The structure of the immunochromatographic test strip comprises a sample pad, an NC membrane and absorbent paper assembled together on a bottom plate.

6. The antigen immunochromatographic detection kit according to claim 5, characterized in that: A detection line and a quality control line are set on the NC membrane, and the concentration of the S100A12 capture antibody coated on the detection line is 0.5-1.5 mg / mL; The quality control line was coated with mouse anti-rabbit IgG at a concentration of 0.5-1.5 mg / mL.

7. The method for preparing the antigen immunochromatographic detection kit according to any one of claims 1 to 6, characterized in that: The method comprises combining the complex solution, a quantum dot-labeled antibody conjugate and an immunochromatographic test strip.

8. Use of the antigen immunochromatographic detection kit according to any one of claims 1 to 6 in the preparation of an early diagnostic reagent for acute myocardial infarction.

9. A method for detecting S100A12 for non-disease diagnosis and / or treatment purposes, characterized in that: When the antigen immunochromatographic detection kit according to any one of claims 1 to 6 is used for detection, the method comprises the following steps: diluting the quantum dot-labeled antibody conjugate with a reconstitution solution, mixing and incubating the conjugate with the sample to be tested to obtain an incubation solution; The incubation solution is spotted onto an immunochromatographic test strip, and the content of S100A12 in the sample to be tested is calculated based on the signal intensity.

10. The S100A12 detection method according to claim 9, characterized in that: The relationship between the signal intensity and the concentration of S100A12 in the sample includes: y = 183.3 + (11389 - 183.3) / (1 + 10 (2.847-x)×1.167 ), R 2 =0.9942, x is the Log value of the S100A12 concentration in the sample, the unit of the S100A12 concentration in the sample is pg / mL, and y is the fluorescence signal value.

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